Related ArticlesMonitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR.
Pharm Res. 2015 Oct;32(10):3365-75
Authors: Aubin Y, Hodgson DJ, Thach WB, Gingras G, Sauvé S
Abstract
PURPOSE: Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is marketed under the brand name Neupogen® by Amgen. Since this product has lost patent protection, many biosimilar versions have been approved or are in the process of filing for market authorization throughout the world. Here we show that NMR spectroscopy can be used to assess the three-dimensional structure of the active ingredient in the formulated approved product Neupogen®.
METHODS: Recombinant metHuG-CSF was prepared in E. coli and isotopically enriched with (13)C and (15) N isotopes. NMR spectroscopy was used to study the effects of excipients on the conformation.
RESULTS: The effects of pH variation on the amide chemical shifts suggest the presence of cation-pi interactions between His-79 and Trp-118, and His-156-Trp-58-His-52 that stabilizes the conformation at low pH. This may be associated with a small local conformational change. The NMR data showed that polysorbate does not interact significantly with filgrastim thus allowing the collection of spectra in the presence of 20 times the formulation concentration in the sample. However, at higher detergent concentrations a reduction of signal intensity is observed. Conclusions The NMR fingerprint assay applied to filgrastim (Neupogen® and a CRS from the European Pharmacopeia (EP)) provided residue specific information of the structure of the drug substance. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate comparability exercises.
[NMR paper] On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.
On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.
Related Articles On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.
Proteins. 2015 Mar 4;
Authors: Sharp KA, O'Brien E, Kasinath V, Wand AJ
Abstract
Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the...
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[NMR paper] Determination of protein structures consistent with NMR order parameters.
Determination of protein structures consistent with NMR order parameters.
Related Articles Determination of protein structures consistent with NMR order parameters.
J Am Chem Soc. 2004 Jul 7;126(26):8090-1
Authors: Best RB, Vendruscolo M
Order parameters obtained from NMR experiments characterize distributions of bond vector orientations. Their interpretation, however, usually requires the assumption of a particular motional model. We propose a multiple-copy simulation method in which the experimental order parameters are used as restraints in...
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[NMR paper] Temperature dependence of NMR order parameters and protein dynamics.
Temperature dependence of NMR order parameters and protein dynamics.
Related Articles Temperature dependence of NMR order parameters and protein dynamics.
J Am Chem Soc. 2003 Sep 17;125(37):11158-9
Authors: Massi F, Palmer AG
The helical subdomain, HP36, of the F-actin-binding headpiece domain of chicken villin, is the smallest naturally occurring polypeptide that folds to a thermostable compact structure. Unconstrained molecular dynamics simulations and constrained molecular dynamics simulations using umbrella sampling are used to study the...
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[NMR paper] Correlation between 2H NMR side-chain order parameters and sequence conservation in g
Correlation between 2H NMR side-chain order parameters and sequence conservation in globular proteins.
Related Articles Correlation between 2H NMR side-chain order parameters and sequence conservation in globular proteins.
J Am Chem Soc. 2003 Jul 30;125(30):9004-5
Authors: Mittermaier A, Davidson AR, Kay LE
Side-chain 2H NMR relaxation data have been collected for the SH3 domain from the Fyn tyrosine kinase and analyzed with respect to sequence preference and per-residue solvent accessibility. Residues that are highly preferred at a given...
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[NMR paper] The effects of mutations on motions of side-chains in protein L studied by 2H NMR dyn
The effects of mutations on motions of side-chains in protein L studied by 2H NMR dynamics and scalar couplings.
Related Articles The effects of mutations on motions of side-chains in protein L studied by 2H NMR dynamics and scalar couplings.
J Mol Biol. 2003 Jun 6;329(3):551-63
Authors: Millet O, Mittermaier A, Baker D, Kay LE
Recently developed 2H spin relaxation experiments are applied to study the dynamics of methyl-containing side-chains in the B1 domain of protein L and in a pair of point mutants of the domain, F22L and A20V. X-ray and...
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[NMR paper] Contact model for the prediction of NMR N-H order parameters in globular proteins.
Contact model for the prediction of NMR N-H order parameters in globular proteins.
Related Articles Contact model for the prediction of NMR N-H order parameters in globular proteins.
J Am Chem Soc. 2002 Oct 30;124(43):12654-5
Authors: Zhang F, Brüschweiler R
An analytical relationship is presented for the estimation of NMR S2 order parameters of N-HN vectors of the protein backbone from high-resolution protein structures. The relationship solely depends on close contacts of the peptide plane to the rest of the protein. Application of the...
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[NMR paper] Long-distance effects of site-directed mutations on backbone conformation in bacterio
Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of Val-labeled proteins.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-cellhub.gif http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of Val-labeled proteins.
Biophys J. 1999 Jul;77(1):431-42
Authors:...
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Script to obtain order parameters from a structure
A Python script for prediction of order paramter from a structure is available from this website.
The script is based on the following paper
F. Zhang and R. Brüschweiler (2002) "Contact Model for the Prediction of NMR N-H Order Parameters in Globular Proteins" J. Am. Chem. Soc. 124(43), 12654-12655.
Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR.
Linear Mode
